The overall theme of this competitive renewal application is to characterize immune responses induced upon gene transfer by adeno-associated virus (AAV) vectors and to devise avenues to circumvent effector Immune responses that impede sustained gene transfer. In its 1(r)'objective, the program will analyze T cell responses to the capsid antigens of AAV vectors pseudotyped with capsids of different serotypes or with genetically modified capsids using in vitro systems, experimental animals and samples from human subjects to assess functionality of T cells induced by natural infections or AAV gene transfer and to determine the longevity of T cell responses to the latter. Avenues to circumvent destructive T cell responses to AAV gene transfer including capsid modifications and pharmacological interventions will be explored. In its 2"" objective, the program will analyze T cell responses to the transgene product of AAV vectors with focus on double-stranded AAV vectors that according to preliminary data of the program elicit more potent innate and adaptive immune responses than single-stranded AAV vectors. In its 3"^ objective, the program will explore the use of regulatory T cells (Tregs) to suppress destructive immune responses against AAV capsid or therapeutic proteins such as clotting factors to prevent a primary response to gene transfer or to down-regulate an existing response to AAV-mediated gene transfer or protein therapy. The program is divided into 3 Projects supported by 2 Cores. Project 1 (KA High): Immune Responses to Capsid in AAV-Mediated Gene Transfer Project 2 (HC ErtI): T Cells to AAV and AAV-Encoded Transgene Products Project 3 (RW Herzog, C Terliorst): Pathways Towards Immune Tolerance to Coagulation Factors Core A (HC ErtI): Administrative Core Core B (S Zliou): Vector Core